Assessment of swab transport systems for aerobic and anaerobic organism recovery.

Saline suspensions of 11 aerobes and anaerobes were used to inoculate swabs from Port-A-Cul (Becton Dickinson), Culturette EZ (Becton Dickinson), and Copan Amies gel (Copan Diagnostics). Swabs were removed from transport devices at 0, 24, and 48 h postinoculation and then extracted by vortexing in 1.0 ml of saline, and organism survival was determined by quantitative plate counts. For the organisms tested, Culturette EZ allowed < 1% of the original inocula to be recovered after 24 h. Port-A-Cul was only slightly better. Recovery was best with the Copan gel-containing system. Agar gel swab systems may be useful for multipurpose transport devices.     

Recovery of anaerobic, facultative, and aerobic bacteria from clinical specimens in three anaerobic transport systems.

With aspirated specimens from clinical infections, we evaluated the recovery of anaerobic, aerobic, and facultative bacteria in three widely used transport systems: (i) aspirated fluid in a gassed-out tube (FGT), (ii) swab in modified Cary and Blair transport medium (SCB), and (iii) swab in a gassed-out tube (SGT). Transport tubes were held at 25 degrees C and semiquantitatively sampled at 0, 2, 24, and 48 h. Twenty-five clinical specimens yielded 75 anaerobic strains and 43 isolates of facultative and 3 of aerobic bacteria. Only one anaerobic isolate was not recovered in the first 24 h, and then, only in the SGT. At 48 h, 73 anaerobic strains (97%) were recovered in the FGT, 69 (92%) in the SCB, and 64 (85%) in the SGT. Two problems hindered the recovery of anaerobes in the SCB and SGT systems: first die-off of organisms, as evidenced by a decrease in colony-forming units of 20 strains (27%) in the SCB and 25 strains (33%) in the SGT, as compared with 7 strains (9%) in the FGT, over 48 h; and second, overgrowth of facultative bacteria, more frequent with SCB and SGT. The FGT method was clearly superior at 48 h to the SCB and SGT systems in this study and is recommended as the preferred method for transporting specimens for anaerobic culture.     

Comparison of two systems for identification of anaerobic bacteria

The RapID Ana and the API 20A systems for identification of anaerobic bacteria were compared for accuracy on 108 recent isolates of gram-negative and gram-positive anaerobic bacteria. No additional tests or gas-liquid chromatography were used. RapID Ana identified 91.7 % of the isolated strains to species level and 6.5 % to genus level but failed to identify 1.8 % of the total strains. API 20A identified 85.2 % of strains to species level, 3.7 % to genus level but failed to identify 11.1 % of the total strains. Although the difference between the identification rates of the two systems was not significant, it was concluded that RapID Ana was easier to perform and interpret.     

Survival of anaerobic and aerobic bacteria on cotton swabs in three transport systems.

The capacity of aerobic and anaerobic bacteria to survive on cotton swabs placed into a dry gassed-out CO2-filled tube (DGT), dry sterile aerobic tube (DAT), and a tube containing a modified Stuarts' transport medium (MST), was assessed. Pseudomonas aeruginosa increased in numbers by 2 and 3 logs when stored in MST and DAT, respectively. The viability of P. aeruginosa, although retarded when compared to MSA and DAT, was not adversely affected by the CO2 environment in the DGT. The MST maintained relatively constant numbers of Streptococcus pyogenes during the 48-h storage period. The DAT and the DGT were unable to maintain the viability of S. pyogenes. Staphylococcus aureus, when stored in a DGT, DAT, or MST, was maintained in relatively constant numbers throughout the entire storage period. Of the four anaerobic bacteria evaluated (Bacteroides fragilis ssp. thetaiotaomicron, Bacteroides melaninogenicus ssp. asaccharolyticus, Fusobacterium nucleatum, and Peptostreptococcus anaerobius), only B. fragilis ssp. thetaiotamicron survived the 48-h storage period in the DGT. Under these test conditions the DGT did not adequately maintain the viability of the majority of anaerobic bacteria tested (when held on cotton swabs). However, the MST did maintain the viability of all species tested for at least the first 2 h of storage.     

Comparison of Easy-Flow Copan Liquid Stuart's and Starplex Swab transport systems for recovery of fastidious aerobic bacteria

Because samples are frequently submitted on swabs from distant sites, viability of the organism must be maintained. We compared two transport systems, a new Copan Liquid Stuart's swab with an Easy-Flow swab applicator and the Starplex Liquid Stuart's swab. The purpose of the study was to assess the release and/or recovery of organisms from the Copan system compared to that from Starplex. Triplicate swabs were seeded with 3 dilutions of Neisseria gonorrhoeae, Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae. Although the amount of the initial inoculum was the same for both transport systems, recovery by the roll-plate method at time zero was consistently increased with the Copan system (31 to 87% higher). This is the most important finding in this study. With N. gonorrhoeae, subsequent recoveries were similar for Copan and Starplex but poor for both systems. With N. meningitidis and Haemophilus, higher levels of recovery were clearly obtained with Copan (P < 0.05 to P < 0.001). With Streptococcus, subsequent recoveries for Copan and Starplex were mixed. In conclusion, Copan generally demonstrated better recovery of organisms compared to Starplex even (and especially) at time zero.     

The aerobic and anaerobic bacteriology of perirectal abscesses.

The microbiology of perirectal abscesses in 144 patients was studied. Aerobic or facultative bacteria only were isolated in 13 (9%) instances, anaerobic bacteria only were isolated in 27 (19%) instances, and mixed aerobic and anaerobic flora were isolated in 104 (72%) instances. A total of 325 anaerobic and 131 aerobic or facultative isolates were recovered (2.2 anaerobic isolates and 0.9 aerobic isolates per specimen). The predominant anaerobes were as follows: Bacteroides fragilis group (85 isolates), Peptostreptococcus spp. (72 isolates), Prevotella spp. (71 isolates), Fusobacterium spp. (21 isolates), Porphyromonas spp. (20 isolates), and Clostridium spp. (15 isolates). The predominant aerobic and facultative bacteria were as follows: Staphylococcus aureus (34 isolates), Streptococcus spp. (28 isolates), and Escherichia coli (19 isolates). These data illustrate the polymicrobial aerobic and anaerobic microbiology of perirectal abscesses.     

Recovery of aerobic and anaerobic bacteria from irradiated mice.

Anaerobic and aerobic bacteria were often found as mixed infections in 225 lethally irradiated mice. Of a total of 57 mice that were sacrificed, aerobic bacteria were recovered exclusively in 9 (27%) of the 34 culture-positive mice, anaerobic bacteria were recovered exclusively in 15 (44%), and mixed aerobic and anaerobic flora were recovered in 10 (29%). The predominant organisms were anaerobic cocci Escherichia coli, Proteus mirabilis, Staphylococcus spp., and Bacteroides spp.     

Survival of anaerobic and aerobic bacteria in a nonsupportive gassed transport system.

Survival of anaerobic and aerobic bacteria in a commercially available, non-supportive, gassed (oxygen-free) transport container (Anaport) was evaluated quantitatively. Saline-suspended obligate anaerobes survived significantly better in the gassed container in aerobic control tubes (P less than 0.025, t test), and counts were virtually unchanged after 8 h of holding. Similarly, initial counts and relative proportions of a mixture of Bacteroides fragilis and Staphylococcus aureus were maintained for 72 h. The value of the gassed transport system was less apparent when microorganisms were suspended in nutrient broth. The major advantage of the gassed transport system appears to be for holding of specimens collected by saline irrigation.     

Isolation of capsulate anaerobic bacteria from orofacial abscesses

The presence of capsulate Bacteroides spp. and anaerobic gram-positive cocci was investigated in pus specimens from 182 children with chronic orofacial infections or abscesses and in pharyngeal swabs from 26 children without inflammation. Of 216 Bacteroides spp. and anaerobic cocci isolated from clinical infections, 170 (79%) were capsulate, compared with 34 (35%) of 96 isolates from normal pharyngeal flora (p less than 0.001). The commonest organisms found to be capsulate more often from infected patients than from controls belonged to the B. melaninogenicus group. The possible evolution of encapsulation in these organisms and their importance in mixed orofacial infections are discussed.     

Comparison of lysis-centrifugation with a biphasic blood culture medium for the recovery of aerobic and facultatively anaerobic bacteria

The Du Pont Isolator tube and Roche Septi-Chek blood culture bottle employ solid media which facilitate the removal of bacteria from static or cidal substances in blood to increase recovery and decrease detection time. In a comparison of 11,567 blood culture sets, the Isolator tube and vented Roche Septi-Chek bottle were positive for 533 (80%) and 494 (74%) of the aerobic and facultatively anaerobic organisms recovered, respectively. This difference was not significant. A significant difference was found in the overall detection time. The Isolator tube recovered the bacteria ca. 1 day earlier. The earlier detection time was most notable with Staphylococcus aureus, viridans streptococci, and Pseudomonas aeruginosa. Among the 355 bacteremic episodes analyzed by a computer program, the Isolator tube was responsible more often for the first report of bacteremia in a given patient. Both systems performed well for the recovery of aerobic and facultatively anaerobic bacteria, but it is recommended that either be used in combination with an unvented broth-containing bottle.     

Influence of the collection and transport of specimens on the recovery of bacteria from peritonsillar abscesses.

In 30 patients with peritonsillar abscesses, pus was obtained by aspiration and by taking a swab after incision; bacterial recovery was compared. Although processed in the laboratory within 2 h, swab speciments gave results comparable to syringe specimens in only 9 of 13 patients with beta-hemolytic streptococci and 7 of 25 patients with anaerobic bacteria. Both kinds of microorganisms were lost in some cases but appeared as additional flora in others. The poor results from the swab technique was ascribed to overgrowth of respiratory flora contaminating the sample after incision. In aspirated pus kept in the syringe, or transferred to anaerobic transporters, the microbial flora was unchanged for 24 to 48 h. Some anaerobes also survived on agar slants for 24 h, but specially designed anaerobic transporters are recommended.     

Evaluation of amniotic fluid for aerobic and anaerobic bacteria.

Studies of 117 pregnant women, 83 at term, were instituted to determine the bacteriologic state of amniotic fluid, utilizing both standard aerobic and anaerobic technics. A high association of postpartum infection was found in women who had long periods of premature reptured membranes and many vaginal examinations. Significant organisms including anaerobes, were isolated in many of these instances. Based on the findings of these studies, it is recommended that amniocentesis for aerobic and anaerobic cultures be done when membranes have been ruptured for 8 hours or more, and when the patient has had seven or more vaginal examinations during the course of labor.     

Comparison of three transport systems for recovery of aerobes and anaerobes from wounds.

A clinical evaluation of three commercially available transport systems (Anaerobic Specimen Collector, Becton-Dickenson; Anaswab, Scott Laboratories; and Trans-Cul, Wampole) was done, using as subjects 75 patients suspected of having infections. Holding times were purposely varied and ranged from 30 min to 48 hours. Totals of 22 aerobic species and 15 anaerobic species were isolated. There was essentially no difference among the three systems in the recoveries of aerobic or anaerobic organisms. Discrepancies appeared to be related to the order of collection and amount of clinical material available for culture. Time delay from collection to plating did not affect the recovery of aerobes or anaerobes. The three systems performed equally in the recovery of clinically significant pathogens from purulent clinical material.     

Comparison of recovery of blood culture isolates from two BacT/ALERT FAN aerobic blood culture bottles with recovery from one FAN aerobic bottle and one FAN anaerobic bottle

Traditionally, a routine blood culture for adult patients consisted of paired aerobic and anaerobic bottles, but the routine use of an anaerobic blood culture bottle has been challenged in recent years. In this study, we compared the recovery of two FAN aerobic bottles with one FAN aerobic and one FAN anaerobic bottle. Each pair of bottles was collected by a separate collection procedure, and each bottle held a recommended 8- to 12-ml draw. A total of 704 clinically significant isolates were recovered from 8,620 sets (17,240 pairs), with 487 (69.2%) isolates recovered from one or both bottles in each pair of bottles, 86 isolates (12.2%) recovered only from the FAN aerobic-FAN aerobic pair, and 131 isolates (18.6%) recovered only from the FAN aerobic-FAN anaerobic pair. Significantly more total organisms (P = 0.002), gram-positive cocci (P = 0.03), Staphylococcus aureus (P = 0.05), Enterobacteriaceae other than Escherichia coli (P = 0.02), and anaerobes (P = 0.01) were recovered from the FAN aerobic-FAN anaerobic pair than from the FAN aerobic-FAN aerobic pair. A separate analysis was performed on the 618 isolates that were recovered from the FAN aerobic-FAN anaerobic pair to compare recovery by bottle type. Significantly more S. aureus (P = 0.005) and anaerobes (P < 0.001) were recovered from the FAN anaerobic bottle, while significantly more coagulase-negative staphylococci (P = 0.01), Streptococcus pneumoniae (P = 0.03), and other gram-negative bacilli (P = 0.004) were recovered from the FAN aerobic bottle. These results support the routine use of a FAN anaerobic bottle for use in the culture of blood with the BacT/ALERT system in our institution. These results also suggest that the decision of whether to routinely utilize an anaerobic blood culture bottle should be influenced by the overall recovery of bacteria and yeast, the recovery of specific types of bacteria or yeast, the medium type, and the blood culture system utilized by the laboratory.     

Evaluation of two single-plate incubation systems and the anaerobic chamber for the cultivation of anaerobic bacteria.

Three systems that are available for the incubation of anaerobic organisms were evaluated to assess their ability to support the growth of 25 anaerobic stock strains and to successfully recover anaerobic bacteria from clinical specimens. These were the anaerobic chamber, the Anaerobic Pouch System Catalyst-Free (Difco Laboratories, Detroit, Mich.), and the Bio-Bag Environmental Chamber Type A (Marion Scientific, Div. Marion Laboratories, Inc., Kansas City, Mo.). Three study centers were involved, the Wadsworth Anaerobe Laboratory (Los Angeles, Calif.), the Good Samaritan Hospital (San Jose, Calif.), and the Massachusetts General Hospital (Boston). A total of 171 anaerobic organisms were isolated from 49 clinical specimens that were cultured at the three test centers. Of these, 169 (99%) were recovered from media that were incubated in the anaerobic chamber, 163 (95%) were recovered from the Anaerobic Pouch, and 147 (86%) were recovered from the Bio-Bag. A similar trend was seen with the stock strains, in which the anaerobic chamber often supported better growth of the organisms than did either of the bag systems.     

Quantitative survival of aerobic and anaerobic microorganisms in Port-A-Cul and Copan transport systems

Transport media should preserve the viability and stability of microorganisms in clinical specimens. In this study, the Port-A-Cul transport system and the Copan transport system without charcoal, both designed to preserve anaerobes, were evaluated. Dacron swabs were inoculated with two combinations of facultative and anaerobic organisms typically found in vaginal swab samples. Combination I contained Candida albicans, Escherichia coli, Enterococcus spp., group B streptococci, Lactobacillus crispatus, and Staphylococcus aureus. Combination II contained Lactobacillus iners, Peptoniphilus asaccharolyticus, Mycoplasma hominis, Prevotella bivia, Prevotella corporis, Porphyromonas asaccharolytica, Mobiluncus curtisii, Peptostreptococcus anaerobius, and Gardnerella vaginalis. Duplicate swabs were placed into the two transporters and held for 24, 48, 72, and 96 h at 4 and 24°C. Both transporters maintained the viability of organisms better at 4°C than at 24°C. Prevotella bivia and Prevotella corporis had a loss of viability in both transporters at both temperatures. However, at 24°C, there was a significantly greater loss of viability for Mycoplasma hominis, Prevotella bivia, Prevotella corporis, and Peptoniphilus asaccharolyticus when the organisms were stored in Copan transport medium than when they were stored in Port-A-Cul transport medium for 96 h (P < 0.002). Some organisms proliferated in the transport media, but when transporters were held at 24°C for 96 h, a significantly greater increase in the concentrations of group B streptococci and Candida albicans, Escherichia coli, and Enterococcus spp. organisms in Copan medium than in Port-A-Cul medium was observed (P < 0.002). At room temperature, the Port-A-Cul system is superior to the Copan system with respect to the preservation of fastidious microorganisms and the prevention of the proliferation of facultative organisms.     

A transport method for swab specimens submitted for aerobic and anaerobic bacteriology.

The need for separate swab transport methods for aerobes and anaerobes may result in inadequate transport of specimens for anaerobic bacteriology. Most microbiology laboratories in Australia rely on Stuart's transport medium to protect anaerobic bacteria. This paper presents a new, simple transport medium (Transport Deep) suitable for sue with aerobes and anaerobes. Comparative evaluations demonstrate that Transport Deep is as good as Stuart's medium for the maintenance of fastidious bacteria and is far superior for the protection of even extremely oxygen-sensitive anaerobes. This medium has been used successfully in a large Sydney hospital for more than a year. It is proposed that Transport Deep be used on a routine basis for all swab specimens.     

Comparison of four commercial microdilution systems for susceptibility testing of anaerobic bacteria

The reliability of four commercially available broth microdilution systems (ATB ANA, MHK-Anaerob-Biotest, Dynatech MIC system, Sceptor Anaerobe MIC system) for routine susceptibility testing was evaluated using agar dilution and broth macrodilution as reference methods. Using the categories susceptible, moderately susceptible and resistant, the rate of essential agreement (complete agreement plus minor errors) of all four systems compared to the two reference methods was satisfactory, ranging from 93% (Dynatech system and agar dilution) to 98 % (Sceptor system and agar dilution). The rate of complete agreement compared to the agar dilution and broth dilution method respectively was 61 % and 60 % for the MHK-Anaerob-Biotest, 65 % and 63 % for the Dynatech system, 72 % and 72 % for ATB ANA, and 85 % and 80 % for the Sceptor system. The Sceptor system was thus superior to the other systems. The systems were easy to operate and inoculate, although arrangement of antimicrobial agents in the Dynatech panels was apt to be confusing.     

Role of anaerobic flora in the translocation of aerobic and facultatively anaerobic intestinal bacteria.

It is thought that the normal enteric microflora acts not only to prevent intestinal colonization but also to prevent subsequent systemic dissemination of ingested, potentially pathogenic bacteria. To determine the relative roles of specific components of the intestinal bacterial flora in bacterial translocation out of the gut, mice were given various antimicrobial agents to selectively eliminate specific groups of intestinal bacteria. The cecal flora and the translocating bacteria in mesenteric lymph nodes were monitored both before and after oral inoculation with antibiotic-resistant Escherichia coli C25. Orally administered streptomycin selectively eliminated cecal facultative gram-negative bacilli, orally administered bacitracin-streptomycin eliminated all cecal bacterial species except low numbers of aerobic sporeformers, and parenterally administered metronidazole selectively eliminated cecal anaerobic bacteria. Compared with control mice, only metronidazole-treated mice had significantly increased rates of dissemination of intestinal bacteria into mesenteric lymph nodes, indicating that the exclusive absence of anaerobic bacteria facilitated the translocation of the intestinal facultative bacteria. In a parallel experiment with streptomycin-resistant E. coli C25 as a marker, parallel results were obtained. Metronidazole increased the translocation of the marker strain and the indigenous strains of intestinal bacteria. Thus, anaerobes appeared to play a key role in confining indigenous bacteria to the gut. However, intestinal colonization and translocation of E. coli C25 occurred most readily after bacitracin-streptomycin treatment, suggesting that in addition to anaerobic bacteria, other bacterial groups may play a role in limiting the intestinal colonization and extraintestinal dissemination of E. coli C25.